The objective of the project is to uncover the mechanisms that govern alternative cell fates of biofilm- matrix production and spore formation in the bacterium Bacillus subtilis. We propose to explain how the interplay among stochasticity, environmental cues and internal regulatory logic governs the choices between alternative programs of cellular differentiation. The proposal addresses important gaps in our understanding of biofilm formation and sporulation with three specific aims: (1) Determine how differential activation of a major regulatory protein controls the cell fates of matrix production and sporulation using a microfluidic platform that enables us to visualize decision-making in individual cells over time. (2) Determine how a sporulation-specific transcription factor is activated in a cell- specific manner and is then suppressed. (3) Determine how matrix production is regulated, both by a dedicated repressor that integrates multiple cues in the decision to form matrix-producing cells and by the exopolysaccharide component of the matrix, which regulates its own synthesis. Research into gene control by B. subtilis, the principal model organism for Gram-positive bacteria, has provided, and will continue to provide, fundamental insights into the molecular and cellular biology of related, pathogenic bacteria, such as Clostridium difficile, Staphylococcus, Enterococcus and B. anthracis.